This research proposal describes studies designed to answer three interdependent questions concerning the mechanism of action of the enzymes which catalyze phospho-ribosyl transfers (PRT) in yeast. 1) What are the active site geometries of the enzymes adenine-, orotatate-, and nicotinate-phosphoribosyltransferase and PRPP-amidotransferase, which catalyze the stereospecific synthesis of beta-nucleotides containing adenine, orotate, nicotinate and an amino group at the 1'-ribose position respectively. 2) What are the binding affinities and conformations of the enzyme-bound substrate phosphoribosyl pyrophosphate (PRPP) and those of the sugar-phosphate inhibitors of these reactions. 3) What is the role in these catalyses of the divalent metal activates. This crucial information on the enzymes, purified from a single source, will allow an evaluation of the similarities and differences in their sugar-binding properties, and provide insight into the substrate-mediated control of several biosynthetic pathways which make use of a common PRPP precursor. The elucidation of potent sugar-phosphate inhibitors of PRT should aid in the design of chemotherapeutic reagents since these enzymes are known to exist in elevated levels in malignant tissue and are deficient in certain metabolic diseases. These investigations will make use of nuclear magnetic resonance and electron paramagnetic resonance techniques which the principal investigator has used recently to determine the conformation and binding affinities of several enzyme-bound nucleotidyl substrates.